Members of the p53 tumor suppressor family (p53, p63 and p73) play critical roles in cell function, animal development and tumor suppression. P63 in particular is known to be crucial for the development of the epidermis. p63 mRNAs can be transcribed through two promoters to produce different N-termini that determine whether they contain (TA) or lack (DN) a full activation domain. TAp63 has been recently proposed to direct keratinocyte commitment during embryogenesis, while DNp63 is the dominant isoform found in both normal and neoplastic keratinocytes with high proliferative potential. Since the molecular mechanisms underlying p63 functions are still largely unknown, we propose to study this by the following approaches. First, we have performed exhaustive yeast two hybrid screening using a human keratinocyte cDNA library and have identified 4 novel p63 interactive partners. We are currently investigating biological functions that are mediated by p63 and its binding partners. Second, the cross talk between p63 and its family members (p53 and TAp63) will be investigated. It has been found that DNp63, the major p63 isoform in keratinocytes, can suppress the activity of p53 and TAp63 in reporter assays. We plan to examine whether DNp63 can inhibit the activity of p53 and TAp63 in vivo, and whether such activity is regulated by the p63-bidning proteins. Third, we will search for direct target genes bound by p63 in keratinocytes using both chromosome immunoprecipitation protocol and DNA microarray analysis. Once those genes are identified, how p63 (together with its binding proteins) regulates them and what their roles in keratinocytes will be further pursued.